Ubiquitin-like proteins. Signal transduction pathways rely on reversible chemical modifications to relay information within and across cells. Covalent modification of protein substrates by ubiquitin and the ubiquitin-like proteins such as SUMO (small ubiquitin-like modifier) contribute to pathways that regulate cellular functions including nuclear transport, cytokinesis, chromosome segregation, G2-M cell cycle progression and transcriptional regulation among many others. Post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins requires the sequential action of E1 activating enzymes, E2 conjugating enzymes and E3 ligases while Ub/Ubl processing and deconjugation is catalyzed by Ub/Ubl-specific proteases. Ubiquitin and SUMO conjugation play an integral role in eukaryotic nuclear metabolism and cell cycle control and our studies are of direct relevance to human health, cancer, and the mission of the NIH. This proposal seeks to address the functional significance for components of the ubiquitin and SUMO conjugation pathways through structural, biochemical and genetic studies that will establish the basis for Ub/Ubl 1) activation, 2) conjugation by E2 and E3 enzymes, 3) and signal transduction through characterization receptors that recognize Ub/Ubl-conjugated substrates. The enzymes, mechanisms and factors that constitute ubiquitin and SUMO protein conjugation pathways are conserved so our studies are broadly relevant to other Ub/Ubl-related pathways. RNA decay. Shifting the balance between RNA transcription and degradation regulates RNA lifetime, quality and abundance. Two principle RNA decay pathways exist in eukaryotes, one catalyzes degradation 5' to 3' while the other degrades RNA in the 3' to 5' direction. The 3' to 5' decay pathway requires the activities of the RNA exosome, a large multi-subunit protein complex that contains a non-catalytic core of nine subunits and two additional subunits that catalyze processive and distributive 3' to 5' RNA exoribonuclease activities. In budding yeast, ten of the eleven genes are essential for life, suggesting the importance of the RNA exosome and its activities in cellular function. Recent efforts illuminated fundamental aspects of eukaryotic exosome structure and function, however many questions remain with respect to the collective activities for exosome subunits in RNA processing and decay and how they are coupled to factors the recruit and modify the RNA substrates. RNA decay pathways play an integral role in eukaryotic nucleic acid metabolism, so our studies are of direct relevance to human health and the mission of the NIH because misregulation of RNA processing and decay is associated with diseases such as cancer, inflammation and neurodegeneration. This proposal will address central issues of human and yeast RNA exosome biology by characterizing individual exosome subunits, by reconstituting multi-subunit RNA exosomes and by analyzing the activities of these complexes in biochemical, genetic and structural studies that will establish functions for the exosome during RNA processing and degradation in vitro and in vivo.